Physiological active substances such as various hormones, neurotransmitters, etc. regulate the biological function via specific receptor proteins present on cell membranes. Many of these receptor proteins are coupled with guanine nucleotide-binding protein (hereinafter sometimes simply referred to as G protein) and mediate the intracellular signal transduction via activation of G protein. These receptor proteins possess the common structure containing seven transmembrane domains and are thus collectively referred to as G protein-coupled receptor proteins (GPCR) or seven-transmembrane receptor proteins (7TMR).
G protein-coupled receptor proteins are present on the cell surface of each functional cell and organ in the body, and play a vitally important physiological role as the target of the molecules that regulate the functions of the cells and organs, e.g., physiologically active substances such as peptide hormones, nucleic acids, amines, lipids, etc. Receptor proteins transmit signals into cells via binding with physiologically active substances, and the signals induce various reactions such as activation and inhibition of the cells.
To clarify the relationship between substances that regulate complex biological functions in various cells and organs in the body, and their specific receptor proteins, in particular, G protein-coupled receptor proteins will elucidate the functional mechanisms in various cells and organs in the body to provide a very important means for development of drugs closely associated with these functions.
For example, in various organs, their physiological functions are controlled in vivo through regulation by many hormones, hormone-like substances, neurotransmitters or physiologically active substances. In particular, physiologically active substances are found in numerous sites of the body and regulate the physiological functions through their corresponding receptor proteins. Many unknown hormones, neurotransmitters or other physiologically active substances still exist in the body and, as to their receptor proteins, many of these proteins have not yet been reported. In addition, a lot remains yet to be established if there are subtypes of known receptor proteins or if there are differences depending on species.
It is very important for development of drugs to clarify the relationship between substances that regulate elaborated functions in vivo and their specific receptor proteins. Furthermore, for efficient screening of agonists for and antagonists to receptor proteins in development of drugs, it is required to clarify functional mechanisms of receptor protein genes expressed in vivo and express the genes in an appropriate expression system.
In recent years, random analysis of cDNA sequences has been actively studied as a means for analyzing genes expressed in vivo. The sequences of cDNA fragments thus obtained have been registered on and published to databases as Expressed Sequence Tag (EST). Also, it has been extensively attempted to predict unknown genes from the analysis of genome sequences. However, since many ESTs or genome sequences contain sequence information only, it is difficult to predict their functions.
The amino acid sequence of human-derived GPR39 and the DNA encoding the same are reported (Genomics 1997, December 15; 46(3):426-34, WO2002/61087, US2002/004491, WO2002/39885, WO2002/71928, WO2001/81634, WO2002/79492, WO2002/86443). However, functions of these G protein-coupled receptor proteins and their physiological ligands remain unresolved.
Heretofore, substances that inhibit the binding of G protein-coupled receptor proteins to physiologically active substances (i.e., ligands) or substances that bind and induce signals similar to those physiologically active substances (i.e., ligands) have been utilized for pharmaceuticals as antagonists or agonists specific to these receptor proteins that regulate the biological functions. Therefore, it is a very important means in search for agonists and antagonists that can be targeted for pharmaceutical development to determine specific ligands for G protein-coupled receptor proteins.
However, many G protein-coupled receptor proteins with unknown functions and many so-called orphan receptor proteins in which the corresponding ligands are yet unidentified are present even at this point of time. Thus, search of ligands for G protein-coupled receptor proteins and elucidation of their functions are eagerly awaited.
G protein-coupled receptor proteins are useful in search for a novel physiological active substance (i.e., ligand) using the signal transduction activity as an indicator and in search for agonists and antagonists to the receptor protein. On the other hand, even though no physiological ligand is found, agonists and antagonists to the receptor protein may be prepared by analyzing the physiological action of the receptor protein through inactivation experiment of the receptor protein (knockout animal). Ligands, agonists or antagonists, etc. of these receptor proteins are expected to be utilized as preventive/therapeutic agents or diagnostic agents for diseases associated with dysfunction or hyperfunction of the G protein-coupled receptor proteins.
Furthermore, the decreased or increased functions of G protein-coupled receptor proteins due to genetic aberration of the G protein-coupled receptor proteins in the body often cause some disorders. In this case, the G protein coupled receptor proteins may be used not only for administration of antagonists or agonists for the receptor proteins, but also for gene therapy by introducing the receptor protein gene into the body (or some particular organs) or by introducing the antisense nucleic acid of the receptor protein gene. In this case, information on the base sequence of the receptor protein is essentially required for investigating deletion or mutation on the gene. The receptor protein gene is also applicable as preventive/therapeutic agents or diagnostic agents for diseases associated with dysfunction of the receptor protein.
The present invention relates to determination of ligands for orphan G protein-coupled receptor proteins with unknown functions and use of the G protein-coupled receptor proteins and their ligands. That is, the present invention aims at providing a method of screening a compound (an antagonist or agonist) or its salt that changes the binding property of the ligand to the G protein-coupled receptor protein; a kit for the screening; a compound (an antagonist or agonist) or its salt that changes the binding property of the ligand to the G protein-coupled receptor protein, which is obtainable by using the screening method or the screening kit; a pharmaceutical comprising a compound (an antagonist or agonist) or its salt that changes the binding property of the ligand to the G protein-coupled receptor protein or a compound that changes the expression level of the G protein-coupled receptor protein; and so on.